DESCRIPTION: The broad objective of this proposal is to understand the molecular details of an important defense mechanism (DNA excision repair) against phenotypic changes and mutations in human cells. Such changes are an important etiological factor in cell survival and cancer. DNA insults result from a wide variety of environmental agents, such as ultraviolet (UV) radiation and chemical carcinogens. Since repair of the majority of these insults occurs via nucleotide excision repair, UV radiation and benzolalpyrene diol epoxide (BPDE) will be used as prototype environmental agents for these studies. In addition, repair of bleomycin-induced DNA damage, which mimics damage by ionizing radiation, will be studied to elicit an important alternative (short patch) excision repair mode which requires less processing at the chromatin level. The composition of nascent and mature repair sites in human chromatin will be analyzed by tagging nascent and mature repair sites with nucleotide analogs, which can be used to isolate these regions as protein-DNA complexes. The structure of these sites will be analyzed using accessibility of histone thiols (reflective of unfolded nucleosomes). Secondly, yeast minichromosomes will be used as model chromatin substrates to study efficiency of repair of UV photoproducts in well defined structural domains in both repair proficient and repair deficient yeast cells. These studies will employ a novel Southern blot analysis to follow repair at specific sites in different chromatin locations, including replication origins and transcribed genes, to allow the search for genes involved in nucleosome remodeling during repair. Third, high resolution mapping of DNA damage and repair will be examined in a positioned nucleosome in vitro (consisting of Xenopus b. 5S rDNA). This nucleosome contains a transcription factor (TFIIIA) binding site and will be used as substrate for repair in cell free extracts of Xenopus oocytes. Finally, repair of bleomycin-induced DNA strand breaks (requiring short patch repair) will be assessed in active RNA polymerase I and II genes in repair deficient human cells. The applicant will use this multifaceted approach to examine the role of chromatin structure in DNA damage and its repair with the ultimate goal of understanding this complex process in human cells. Since DNA lesions may alter the expression of specific genes required for establishing the neoplastic phenotype, these studies may provide valuable insight into the cell s defense mechanism for resisting neoplastic transformation by environmental carcinogens.